V(D)J Recombination is a site-specific recombination reaction that occurs in developing lymphocytes. Defective V(D)J recombination is one of the first possibilities in any human immunodeficiencies. Lymphoid specific Recombination Activating Gene 1 and 2 (RAG1 and RAG2) and ubiquitous proteins Ku70, Ku80, DNA-PKcs, Artemis, Ligase IV, XRCC4, Cernunnos/XLF and a number of polymerases catalyze this highly regulated reaction. Human diseases associated with mutations in the V(D)J recombination machinery have been identified for RAGI, RAG2, Artemis, Ligase IV and Cernunnos/XLF. Mutations in these genes lead to different degrees of immune deficiency and can result in Severe Combined Immune Deficiency (SCID), Combined Immune Deficiency (CID) and Omenn's Syndrome. Preliminary studies presented in this application suggest novel molecular mechanisms that undertine RAG2 and Artemis mediated immunodeficiencies. Our studies imply a role for deregulated cellular localization of RAG2 in RAG2 mediated immunodeficiency. Additionally, our data indicate that anomalous junctional diversity plays a major role in Artemis mediated immunodeficiency. Studies using cell lines developed from a subset of patients from the Primary Immune Deficiency Program (PPG) at Mount Sinai School of Medicine point out that some of them carry a novel V(D)J defect. The three aims in this proposal will answer the following important questions: what determines deregulated cellular localization of RAG2 mutants associated with SCID and Omenn Syndrome; what is the impact of nuclease defective Artemis mutants, from SCID and Atypical SCID/Omenn syndrome patients, in generation of junctional diversity at the endogenous lymphocyte receptor genes; and to what extent is defective V(D)J recombination responsible for the immunodeficiencies in patients under study in this PPG? The proposed work will further our current understanding of V(D)J mediated immunodeficiency and might provide critical mechanistic information to aid the development of novel treatment for these life threatening diseases. RELEVANCE (See Instructions): This proposal will analyze the mechanisms that underline V(D)J mediated immunodeficiencies, SCID, CID, Omenn and Atypica SCID/Omenn syndrome, and will also investigate the presence of V(D)J defects in a subset of CVID and yet uncharacterized SCID patients. Results obtained from the proposed study will facilitate the path to finding better treatments for these debilitating diseases.